Puneet Chopra

Role of Mycobacterium tuberculosis Ser/Thr Kinase PknF: Implications in Glucose Transport and Cell Division

Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3H-labeled and 14C-labeled substrates. A significant increase in the uptake of D-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.

Polo-like kinase inhibitors: an emerging opportunity for cancer therapeutics

Importance of the field: The Polo-like kinase (Plk) family has emerged as an important regulator in cell cycle progression. Plks belong to a family of serine/threonine kinases and exist in four isoforms Plk1- 4. However, only one of these isoforms, Plk1, is shown to be involved in the activation of Cdc2, chromosome segregation, centrosome maturation, bipolar spindle formation and execution of cytokinesis. The activity of Plk1 is elevated in tissues and cells with a high mitotic index. In patients, Plk1 is overexpressed in tumors including those derived from lung, breast, colon, pancreas, prostate and ovary. Plk1 depletion is associated with the decrease in cell viability and induction of apoptosis in various cancerous cells. Several Plk1 inhibitors are in different phases of clinical development for anticancer therapy. Areas covered in this review: The focus of present review is to highlight Plk1 as a promising therapeutic approach for the treatment of cancer. The review discusses the role of Plk1 in cancer and the current status of Plk1 inhibitors, as well as highlighting the possible beneficial effect of inhibition of Plk1 as compared to other mitotic targets. What the reader will gain: Readers will get a comprehensive overview of Plk1 as a novel anticancer drug target. This review will also update readers about the progress made in the field of Plk1 inhibitors. Take home message: The current literature about Plk1 inhibitors and knockout studies favor Plk1 inhibition as a potential antitumor therapy.

Recent advances towards identification of new drug targets for Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a very successful pathogen that remains a leading infectious killer worldwide. The global situation has become precarious due to various factors such as the variable efficacy of the Bacille Calmette–Guerin (BCG) vaccine, drug resistance, delay in diagnosis, association with HIV, and other factors, creating a long-lasting reservoir of impending disease and infection. Surprisingly, no new drugs have been developed in the past 30 years. The release of the complete genome sequence of M. tuberculosis and the availability of advanced genetic tools have provided the powerful repertoire of potential drug targets that are now in hand and can be explored in a more rational and directional manner. In this review, the authors highlight some possible therapeutic targets in M. tuberculosis. The gene products involved in various processes, such as mycobacterial cell wall synthesis, ability to acquire or obtain essential nutrients, persistence, transcription regulation, energy metabolism and others, such as the PE-PGRS family and culture filtrate proteins, would be potential targets for the development of new drugs. Apart from these categories, the importance of signal transduction events in the virulence of mycobacteria is discussed in relation to their potential as therapeutic targets. The potential of all of these therapeutic targets should be investigated together with the potential of being able to synthesise future chemotherapeutic agents.

Nucleoside diphosphate kinase of Mycobacterium tuberculosis acts as GTPase-activating protein for Rho-GTPases

Several bacterial pathogens secrete proteins into the host cells that act as GTPase‐activating proteins (GAPs) for Rho‐GTPases and convert GTP‐bound active form to GDP‐bound inactive form. However, no such effector molecule has been identified in Mycobacterium tuberculosis. In this study, we show that culture supernatant of M. tuberculosis H37Rv harbors a protein that stimulates the conversion of GTP‐bound Rho‐GTPases to the GDP‐bound form. Nucleoside diphosphate kinase (Ndk) was identified as this culture supernatant protein that stimulated in vitro GTP hydrolysis by members of Rho‐GTPases. The histidine‐117 mutant of Ndk, which is impaired for autophosphorylation and nucleotide‐binding activities, shows GAP activity. These results suggest that Ndk of M. tuberculosis functions as a Rho‐GAP to downregulate Rho‐GTPases, and this activity may aid in pathogenesis of the bacteria.

Nucleoside diphosphate kinase‐like activity in adenylate kinase of Mycobacterium tuberculosis

Ak (adenylate kinase) is a ubiquitous enzyme that catalyses a reversible high‐energy phosphoryl‐transfer reaction between ATP and AMP to form ADP. In the present study, the Ak gene (adk) of Mycobacterium tuberculosis was cloned, expressed in Escherichia coli and purified as a glutathione S‐transferase fusion protein. Purified Ak converted AMP into ADP in the presence of [γ‐32P]ATP or [γ‐32P]GTP. Replacement of arginine‐88 of adk with glycine resulted in the loss of enzymic activity. The purified protein also showed Ndk (nucleoside diphosphate kinase)‐like activity as it transferred terminal phosphate from [γ‐32P]ATP to all nucleoside diphosphates, converting them into corresponding triphosphates. However, Ndk‐like activity of Ak was not observed with [γ‐32P]GTP. Immunoblot analysis of various cellular fractions of M. tuberculosis H37Rv revealed that Ak is a cytoplasmic protein. The dual activity of Ak as both nucleoside mono‐ and di‐phosphate kinases suggested that this enzyme may have a role in RNA and DNA biosynthesis in addition to its role in intracellular nucleotide metabolism.

Biochemical characterization of an S-adenosyl-l-methionine-dependent methyltransferase (Rv0469) of Mycobacterium tuberculosis.

Abstract Tuberculostearic acid (l0-methylstearic acid, TSA) is a major constituent of mycobacterial membrane phospholipids, and its biosynthesis involves the direct methylation of oleic acid esterified as a component of phospholipids. The methyltransferases of mycobacteria were long proposed to be involved in the synthesis of methyl-branched short-chain fatty acids, but direct experimental evidence is still lacking. In this study, we identified the methyltransferase encoded by umaA in Mycobacterium tuberculosis H37Rv as a novel S-adenosyl-l-methionine (SAM)-dependent methyltransferase capable of catalyzing the conversion of olefinic double bond of phospholipid-linked oleic acid to biologically essential TSA. Therefore, UmaA, catalyzing such modifications, offer a viable target for chemotherapeutic intervention.

Pharmacological profile of AW-814141, a novel, potent, selective and orally active inhibitor of p38 MAP kinase

The p38 mitogen activated protein kinase (MAPK) is a key signaling molecule that plays a crucial role in the progression of various inflammatory diseases such as rheumatoid arthritis (RA), asthma and chronic obstructive pulmonary disease. The objective of the present study was to evaluate the anti-inflammatory activity of a p38 MAPK inhibitor, AW-814141. AW-814141 inhibited enzymatic activity of recombinant p38-alpha and beta isoforms with IC(50) value of 100nM and 158nM, respectively. AW-814141 also inhibited the release of tumor necrosis factor (TNF)-alpha by lipopolysaccharide (LPS) treated human peripheral blood mononuclear cells with an IC(50) value of 212nM and demonstrated selectivity against a panel of few kinases. Oral administration of AW-814141 (10mpk) in LPS-injected mice resulted in a significant reduction in TNF-alpha production in the circulation. In a carrageenan-induced rat paw edema model and collagen-induced arthritis model (CIA), AW-814141 dose dependently inhibited paw swelling. In different in vivo efficacy models, efficacy of AW-814141 was found to be better as compared to the reference compounds (Vx-745 and BIRB-796). This study demonstrated that AW-814141 is a novel p38 MAPK inhibitor and it displays promising in vitro and in vivo anti-inflammatory activities and can be used for the treatment of rheumatoid arthritis.

Cloning and characterization of GTP-binding proteins of Mycobacterium tuberculosis H37Rv

GTP-binding proteins (G-proteins) are highly conserved signaling molecules that participate in cellular signaling and bacterial pathogenesis by regulating the activity of cognate GTPases. However, the exact role of G-proteins in the pathogenesis of Mycobacterium tuberculosis is poorly understood. The complete genome sequence of M. tuberculosis H(37)Rv, suggests the presence of several homologs of bacterial G-proteins. In the present study, three G-proteins, Era, Obg and LepA of M. tuberculosis H(37)Rv were cloned and expressed in Escherichia coli. Purified proteins showed GTP-binding and hydrolyzing activities. A point mutation in the conserved GTP-binding motif, AspXXGly (Asp to Ala) in Era (Asp-258) and Obg (Asp-212) proteins resulted in the loss of the associated activities, confirming that known key residues in well-established G-proteins are also conserved in mycobacterial homologs. This study confirms that Era, Obg and LepA of M. tuberculosis H(37)Rv possess GTPase activity and provide a platform to understand the physiological significance of these proteins in associated pathogenesis.

Development of cell death-based method for the selectivity screening of caspase-1 inhibitors

Caspase-1 selective inhibitors are novel therapeutic agents for inflammatory diseases. Selectivity assays for caspases can be initiated with purified enzyme, making these assays very costly and time consuming. Therefore, there is a need to develop a fast and reliable cell-based assay, which can be used for the selectivity screening of multiple caspases in a biologically relevant context in a single assay. In this study, we have developed an assay in which DNA fragmentation, a hallmark of apoptosis, of Jurkat cell line was examined post induction with etoposide in the presence or absence of inhibitors of caspases 1, 3, 8, 9 and pan-caspase inhibitors. We observed that caspases-3, -8, -9 and pan caspase inhibitors resulted in significant inhibition of etoposide-induced DNA fragmentation. However, caspase-1 specific inhibitor failed to prevent DNA fragmentation, suggesting that either caspases belonging to caspase-1 family (1, 4 and 5) are not present in the Jurkat cells or might not be involved in the etoposide-induced DNA fragmentation. Since the inhibition of caspases 3, 8 and 9 is accompanied by the down regulation of the activity of a cascade of caspases (caspases 2, 6, 7, 9 and 10), selectivity of caspase-I inhibitors can be ascertained for the above panel (caspases 2, 6, 7, 8, 9 and 10) of caspases from this single assay.